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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
  • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
  • C07D213/40—Acylated substituent nitrogen atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6887—Antibody-chelate conjugates using chelates for therapeutic purposes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
  • A61K49/0058—Antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
  • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
  • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
  • C07D213/38—Radicals substituted by singly-bound nitrogen atoms having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
  • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
  • C07D213/42—Radicals substituted by singly-bound nitrogen atoms having hetero atoms attached to the substituent nitrogen atom
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/08—Bridged systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2121/00—Preparations for use in therapy

Definitions

• the present invention relates to novel immunoreagents and more particularly to targeting radioactive immunoreagents which find particular utility in therapeutic and diagnostic imaging compositions and methods.
• the present invention further relates to novel complexing agents.
• radiolabeled-antibodies would concentrate radiolabeled-antibodies to tumor associated antigens and that radiolabeled reagents employed allowed both diagnostic imaging of tumors, e.g., by gamma camera imaging (radioimmunoscintigraphy) and positron tomography, and
• therapeutic treatment i.e., reduction in tumor size by the targeting radioactive immunoreagent.
• gamma radiation of 125 I is too weak for imaging.
• 131 I has often been used but is undesirable because of its long half- life and high energy gamma and cytotoxic beta radiations.
• 131 I has also been used therapeutically for large tumors, but appears ineffective in the treatment of small tumors.
• radioactive iodine exist for diagnostic infarct imaging as for tumor imaging and therapeutic treatment.
• EDTA ethylene diaminetetraacetic acid
• DTPA diethylenetriaminepentaacetic acid
• antibodies for specific targeting antibodies that complex or bind directly with radionuclides, preferred radionuclides and combinations thereof with antibodies and complexing agents.
• Yttrium-90 is an attractive isotope for therapy...but its clinical use will be very limited because of bone marrow toxicity, resulting from acid-promoted release of 90 Y from an antibody linked chelate such as diethylenetriamine-pentaacetic acid (DTPA).
• DTPA diethylenetriamine-pentaacetic acid
• chelating agents When chelating agents are covalently bonded to proteins (such as Ab's), the proteins usually are capable of accepting far more than one molecule of the chelating agent because they contain a host of amine and sulfhydryl groups through which the chelating agents are bound. It is often very important to determine how many chelating sites are bound to each protein molecule. The most convenient way to accomplish this is by spectrophotometric means.
• prior art chelating agents and chelates thereof have spectral absorptions that overlap with those of useful proteins, and an analytical determination, of the number of chelating or chelated sites per molecule of protein cannot be made unequivocally by
• the chelator must be activated by a reducing agent before forming the radionuclide chelate. If the protein conjugates are to be formed prior to formation of the radionuclide
• the reducing agent employed for activating the complexing agent can degrade the protein.
• the preferred chelating agents currently used for complexing technetium (Tc) and rhenium (Re) complex to the metals via sulfur-containing groups which must be reduced with a reducing agent (dithiothreitol) to activate the chelator before forming the radionuclide chelate.
• a reducing agent dithiothreitol
• the reducing agent can degrade the protein . It would be highly desirable to have chelating agents capable of forming conjugates with proteins before complexing with radionuclides.
• radiolabeled antibodies and chelating agents employed for making immunoreactive conjugates by covalently bonding a chelating agent to the immunoreactive protein, and radionuclide complexes thereof for use in diagnostic imaging and targeted therapeutics suffer from one or more of the following
• the targeting radioactive immunoreagents of this invention comprise a metal radionuclide ion, a complexing agent which is a derivative of a pyridine, bipyridine,
• terpyridine quaterpyridine, quinquepyridine, sexipyridine or phenanthroline, and an immunoreactive group covalently bonded through a protein reactive group to the complexing agent.
• a targeting radioactive immunoreagent comprising a metal radionuclide ion, a complexing agent, and an
• R represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, alkylformamido, aryl, aryloxy, heterocyclyl or a protein reactive group
• R 1 represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, alkylformamido, aryl, aryloxy, heterocyclyl or a protein reactive group;
• R 2 represents hydroxy, carboxy, hydroxyalkyl, thioalkyl, carbonyliminodiacetic acid, methyleneiminodiacetic acid, methylenethioethyleneiminodiacetic acid,
• carboxyalkythioalkyl, hydrazinylidenediacetic acid, or a salt of such acids, or two R 2 groups, taken together, represent the atoms necessary to complete a macrocyclic ring structure containing at least one heteroatom coordinating site and at least one, preferably two, alkylene groups forming part of the ring structure;
• R 3 represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, alkylformamido, aryl, aryloxy, heterocyclyl or a protein reactive group;
• R 4 represents hydrogen or a protein reactive group; n is 0, 1, 2, 3 or 4;
• o 0 or 1
• n 0 or 1
• n and m are 0 and at least one of R, R 1 , R 3 and R 4 is a protein reactive group.
• the pyridines have the structure A-II
• R, R 1 , R 2 and R 3 are as defined above and n is 0, 1, 2,
• the phenanthrolines have the structure A-IV
• R 2 , R 3 and R 4 are as defined above.
• This invention provides novel terpyridines, quaterpyridines, quinqepyridines, and sexipyridines having the structure A-I.
• R is , wherein R 5 is alkoxy or alkyl, p is 0, 1, 2, 3 or 4 and R 6 is a protein reactive group.
• This invention further provides novel phenanthrolines preferably having the structure A-IV above wherein at least one R 4 is a protein reactive group.
• This invention also provides therapeutic and diagnostic compositions comprising the above-described
• This invention further provides a method for diagnostic imaging a site in a patient comprising a)
• a radiation-sensitive element or device such as, for example, a film or electronic sensor
• radioactive immunoreagent capable of targeting the site and a pharmaceutically acceptable carrier therefor.
• the described targeting radioactive immunoreagents containing yttrium exhibit lower radiation toxicity, when compared to radioactive immunoreagents prepared with other yttrium
• the targeting immunoreagents of this invention are not rapidly metabolized and do not deleteriously disperse.
• immunoreagents exhibit good emission characteristics and are readily subject to spectrophotometric analysis. Additionally, protein conjugates of the complexing agents can be formed and stored until metal complexation is desired, and complexation can be accomplished without
• the complexing agents rapidly complex with metals, and the resulting chelates exhibit excellent stability with respect to time, temperature and pH.
• FIG. 1 depicts immunocompetency assays of B72.3-THT- Sc +++ , a radioactive immunoreagent of this invention, a B72.3-THT conjugate and unmodified B72.3.
• Fig. 2 depicts immunocompetency assays of a B72.3-THT conjugate, two preparations of B72.3-TMT conjugates and unmodified B72.3.
• Fig. 3 depicts the results of a biodistribution study of B72.3-TMT- 111 In, a radioactive immunoreagent of the
• Fig. 4 depicts the results of a biodistribution study of B72.3-TMT- 90 Y, a radioactive immunoreagent of the invention, and B72.3-DTPA- 90 Y.
• Fig. 5 is a survival curve, i.e., the number of mice surviving each day past the first day of innoculation for a low dose regimen of B72.3-DTEA and a high dose regimen of B72.3-TMT and of B72.3-DTPA.
• Figs. 6 and 7 are absorption spectra of TMT, TMT-Y +++ and TMT-Pb ++ . Description of Preferred Embodiments
• targeting radioactive immunoreagents are useful as diagnostic reagents, for example,
• radioimmunoelectrophoresis reagents
• the immunoreagents of this invention comprise a metal radionuclide ion, a complexing agent, and an immunoreactive group covalently bonded to the complexing agent through a protein reactive group.
• the complexing agent is a derivative of a pyridine, bipyridine, terpyridine, quaterpyridine, quinquepyridine, sexipyridine or phenanthroline, preferably having the
• Each R in formula A-I independently is hydrogen
• alkyl straight or branched alkyl, preferably containing from 1 to about 20 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, s-butyl, t-butyl, 2-ethylhexyl, decyl, hexadecyl, octadecyl, etc.; alkoxy, the alkyl portion of which contains from 1 to about 20 carbon atoms as described for R above;
• alkylthio the alkyl portion of which contains from 1 to about 20 carbon atoms as described for R above
• alkylamino the alkyl portion of which contains from 1 to about 20 carbon atoms as described for R above
• alkylformamido the alkyl portion of which contains from 1 to about 20 carbon atoms as described for R above
• substituted or unsubstituted aryl preferably
• containing from about 6 to 20 carbon atoms such as phenyl, naphthyl, phenanthryl, nitrophenyl, hydroxyphenyl, aminophenyl, hexadecylaminophenyl, octadecylaminophenyl, tolyl, xylyl, methoxyphenyl, 3-amino-4-methoxyphenyl, 4-methoxy-3-(N-methylhydrazinothioformamido)phenyl, 3-isocyanato-4-methoxyphenyl, 3-isothiocyanato-4-methoxyphenyl,
• alkylphenyl the alkyl portion of which contains from 1 to about 20 carbon atoms as described for R above; aryloxy, the aryl portion of which contains from 6 to about 20 carbon atoms as described for R above; a substituted or unsubstituted heterocyclyl, preferably containing 5 or 6 nuclear carbon and heteroatoms such as N, S, P or O, such as pyridyl,
• R is a 4-alkoxy-3-aminophenyl or a 4-alkoxy-3-isothiocyanato phenyl group.
• R 1 is independently selected from the groups specified for R.
• R 1 preferably represents hydrogen or a protein reactive group.
• Each R 2 is independently selected from hydroxy
• hydroxyalkyl the alkyl portion of which preferably contains from 1 to 4 carbon atoms, such as hydroxymethyl;
• methyleneiminodiacetic acid [-CH 2 N(CH 2 COOH) 2 ]; methylenethioethyleneiminodiacetic acid [-CH 2 SCH 2 CH 2 N(CH 2 COOH) 2 ]; carboxyalkythioalkyl, the alkyl portions of which independently contain from 1 to 4 carbon atoms, such as -CH 2 CH 2 SCH 2 CH 2 COOH; 1-hydrazin-2-ylidenediacetic acid, such as 1-hydrazin-2-ylidenediacetic acid,
• heteroatom coordinating site for ions and (b) at least one, preferably two, alkylene groups forming part of the ring structure.
• the macrocyclic ring-forming groups can be a heteroatom group substituted alkylene such as 2,2-bis- (ethoxycarbonyl)-1,3-propylene; or heteroatom-containing groups such as oxybis(alkylene), for example oxybis(ethylene), oxybis(ethyleneoxymethylene), oxybis(ethyleneoxyethylene);
• alkyleneoxyalkyleneoxyalkylene such as methyleneoxyethyleneoxymethylene; arylene-di(oxyalkylene), such as 1,4-dimethyl-5,6-phenylenebis(oxymethylene); 2,6-pyridylenebis(methyleneoxymethylene); 2-methoxy-5-methyl-1,3-phenylenebis(methyleneoxymethylene) and 1,10-phenanthrolin-2,9-ylenebis(methyleneoxymethylene); carboxymethyliminobis[(trimethylene(carboxymethyl)iminomethylene], [-CH 2 N(CH 2 COOH) (CH 2 ) 3 - N(CH 2 COOH)(CH 2 ) 3 N(CH 2 COOH)CH 2 -]; carboxymethylthioethyliminobis [trimethylene (carboxymethyIthioethyl) iminomethylene], [-CH 2 N(CH 2 CH 2 SCH 2 COOH)(CH 2 ) 3 -N(CH 2 CH 2 SCH 2 COOH)
• R 2 is
• R 3 is independently selected from the groups specified for R.
• R 3 preferably represents hydrogen.
• Each R 4 is independently selected from hydrogen or a protein reactive group.
• At least one of the R, R 1 , R 3 and R 4 groups present is a protein reactive group.
• R 4 groups per molecule is a protein reactive group.
• protein reactive group is meant any group which can react with any functional groups typically found on or introduced into proteins. However, it is specifically
• protein reactive group can be conjugated to nonprotein biomolecules.
• protein reactive groups useful in the practice of this invention include those groups which can react with any biological molecule (including
• Preferred protein reactive groups can be selected from, but are not limited to: (1) A group that will react directly with the amine or sulfhydryl groups on the protein or biological molecule containing the immunoreactive group, for example, active halogen containing groups including, for example, chloromethylphenyl groups and chloroacetyl [Cl- CH 2 CO-] groups, activated 2-leaving group substituted
• protein reactive group such as those mentioned in (1) above, for example, by partial oxidation of the protein to introduce an aldehyde or a carboxylic acid, in which case the "protein reactive group” can be selected from amino, alkylamino,
• the alkyl portions of the protein reactive group can contain from 1 to about 20 carbon atoms as described for R above.
• the aryl portions of the protein reactive group can contain from about 6 to about 20 carbon atoms as described for R above.
• a group that can be linked to the protein or biological molecule containing the immunoreactive group, or to the modified protein as noted in (1) and (2) above by use of a crosslinking agent Certain useful crosslinking agents, such as, for example, difunctional gelatin hardeners, bisepoxides and bisisocyanates become a part of, i.e., a linking group in, the protein-complexing agent conjugate during the crosslinking reaction. Other useful crosslinking agents, however,
• crosslinking agents examples include carbodiimide and carbamoyIonium crosslinking agents as disclosed in U.S. Patent 4,421,847, the disclosure of which is hereby incorporated herein by reference in its entirety, and the dication ethers of U.S. Patent 4,877,724, the disclosure of which is hereby incorporated herein by reference in its entirety.
• crosslinking agents one of the reactants must have a carboxyl group and the other an amine or sulfhydryl group.
• crosslinking agent first reacts selectively with the carboxyl group, then is split out during reaction of the "activated" carboxyl group with an amine to form an amide linkage between the protein and metal complexing agents having the structure A-I above, thus covalently bonding the two moieties.
• crosslinking of like molecules e.g., complexing agents with complexing agents
• reaction of difunctional crosslinking agents is nonselective so that unwanted crosslinked molecules are obtained.
• protein reactive groups include amino and isothiocyanate
• Especially preferred complexing agents include species 1-59 set forth below.
• Preferred classes of complexing agents for use herein include terpyridines represented by structure A-III above and phenanthrolines represented by structure A-IV above.
• a particularly preferred class of complexing agents has the structure A-III above wherein n-1 and R is a substituted phenyl containing an alkyl or alkoxy substituent and a protein reactive group.
• Representative preferred species of complexing agents include compounds 20-32 depicted above. The currently most preferred complexing agent is TMT isothiocyanate (compound 24).
• R 6 is a protein reactive group.
• R 5 is alkyl, preferably containing from 1 to about 20, more preferably from 1 to 8 carbon atoms, such as methyl, ethyl and the like; or alkoxy, the alkyl portion of which contains from 1 to about 20, more prefereably from 1 to 8 carbon atoms, such as methoxy, ethoxy and the like.
• R 6 is a protein reactive group as described above. Preferred protein reactive groups include amino, alkylamino, arylamino, carbazido, semicarbazido,
• Especially preferred protein reactive groups include amino, isothiocyanato, and semicarbazido.
• Especially preferred species include TMT (compound 21) TMT isothiocyanate (compound 24), and THT (compound 20).
• Preferred phenanthrolines according to this invention have the structure A-IV above wherein at least one R 4 is a protein reactive group.
• Preferred protein reactive groups include those specified for R 6 above.
• iminodiacetate groups can be prepared by techniques known in the art. Suitable reaction schemes are illustrated in U.S.
• amine groups can be added to phenyl-substituted polypyridines and phenanthrolines by nitration followed by reduction of the nitro groups to amines.
• the amine groups can be readily converted to isocyanate groups by reaction with phosgene to produce the carbamoyl chloride which, upon heating, releases HCl to produce the isocyanate.
• Carboxy groups can be added by treatment of amine-containing
• the presence of the alkyl or alkoxy group on the dibrominated intermediate (63) provides enhanced solubility in THF which is a preferred solvent used in the preparation of the intermediate diol (66).
• TMT (70) is a member of a subclass of the pyridine-containing chelating species of structure A III, namely, the terpyridine system defined by structure (71) wherein R at the 4'-position can be a protein reactive group.
• R at the 4'-position can be a protein reactive group.
• it is a member of a further subclass within (71), which is defined as of the 4'-aryl substituted terpyridine family of compounds (72) wherein either X or Y is a protein reactive group.
• TMT An intermediate in the synthesis of TMT is the tetraester (73) This can be saponified to the tetracarboxylate (74) using sodium hydroxide and then demethylated using HI and red phosphorous to provide the phenol (75).
• Phenol (75 ) can be cyanoethylated with acrylonitrile and then treated with an alcohol such as ethanol in the presence of anhydrous HCl to provide the amidate (76a) .
• the phenol can be alkylated as the phenolate with an alpha-cyano-omega-haloalkane followed by treatment again with alcoholic HCl to provide (76b) .
• Phenol (75) can also be treated with divinyl sulfone to obtain a monoaddition compound (77a) that provides a vinyl sulfone protein reactive group.
• (75) can be alkylated with an omega-hydroxy-alpha-alkyl halide (or sulfonate) or with an omega-hydroxy-alpha-polyalkylenoxyalkyl halide (or
• (75) can be alkylated with an alpha haloacetate ester such as methyl bromoacetate and then treated with hydrazine to provide the hydrazide (78) which is capable of reacting with aldehyde functionality such as, for example, that introduced by oxidation of a carbohydrate moiety attached to an antibody.
• an alpha haloacetate ester such as methyl bromoacetate
• hydrazine to provide the hydrazide (78) which is capable of reacting with aldehyde functionality such as, for example, that introduced by oxidation of a carbohydrate moiety attached to an antibody.
• hydrazide (78) can be treated with an aqueous acid such as HCl and then with sodium nitrite to provide the carbonyl azide (79). This can be reacted with amines on proteins at pH's above seven to provide amide linked chelates.
• phenol (75) can alkylated to produce an alkylene or oxyalkylene spacer group which is terminated with a hydroxyl or protected hydroxyl functional group as in the case of the preparation of the vinyl sulfones (77b).
• the aliphatic alcohol can then be treated with an aryl chloroformate such as p-nitrophenyl chloroformate or 2,4,5-trichlorophenyl
• arylchloroformate can react with amine groups or proteins such as amines in lysines and at peptide amine termini.
• (75) can be reacted directly with cyanuric chloride to produce the cyanurate (81a), or an alcohol, derived from (75) by alkylation, which contains a spacer linkage such as the alcohol intermediate in the preparation of (80) can be so treated to produce the cyanurate (81b).
• chlorocyanurates can react with amine. groups on proteins.
• phenol (75) can be converted to aldehyde (82a) by treatment with acrolein, and to aldehyde (82b) by treatment with a haloalkyl acetal (as an aldehyde protecting group) followed by deprotection of the aldehyde with aqueous acid.
• aldehydes are suitable for reductive amination procedures (e.g., those incorporating the use of sodium cyanoborohydride as the reducing agent at a ph in the range of 4 to 7,
• phenol (75) can be converted to amine (83a) by treatment with acrylonitrile (e.g. in the presence of
• N-methylimidazole as catalyst followed by hydrogenation of the nitrile in warm acetic acid solution using hydrogen and 10 % palladium on carbon as a catalyst.
• (75) can be converted to amine (83b) by alkylation with an omega-haloalkyl nitrile followed by hydrogenation of the nitrile in warm acetic acid solution using hydrogen and 10 % palladium on carbon as a catalyst.
• the above amines can be attached to proteins In several ways. For example, they are suitable for use in reductive amination procedures (e.g., incorporating the use of sodium cyanoborohydride as the reducing agent at a pH of about 6 in an aqueous system) to couple the amine with aldehyde functionality that is generated by the action of an oxidizing agent such as sodium periodate on a carbohydrate moiety attached to a protein (such as an antibody).
• Aldehyde functionality can also be generated on a protein using reagents such as 4-azophenylglyoxal (APG, available from Pierce Chemical Co.) which reacts selectively with arginine residues on the protein.
• APG 4-azophenylglyoxal
• aldehydes can then be reacted with the amines (83a) and (83b) as above.
• these amines can be attached to proteins by first being further elaborated with commonly available heterobifunctional reagents (such as are available commercially from Pierce Chemical Company).
• heterobifunctional reagents such as are available commercially from Pierce Chemical Company.
• the amines represented by (83b) car. be treated with 2-iminothiolane to generate (84a) or with N-succinimidyl S-acetylthioacetate
• the amines represented by (83b) can be treated with SMCC [N-(4-carboxycyclohexylmethyl)-maleimide N-hydroxysuccinimidate] to generate (84c).
• SMCC N-(4-carboxycyclohexylmethyl)-maleimide N-hydroxysuccinimidate
• These species contain maleimide functionality which can react with free sulfhydryl SH groups that can be introduced at amine sites in a
• sulfhydryl-containing reducing agents such as dithiothreitol on disulfide bonds which may be present in the protein or desired protein fragment.
• linking chemistries from amine (85) where X is as defined above.
• Amine (85) can be treated with aqueous hydrochloric acid and sodium nitrite (to form HONO) which will then react with the amine to form the diazonium salt (86).
• This material can be coupled to tyrosine moieties on proteins via diazo linkages to the aromatic ring containing the hydroxyl group. This is represented schematically as (87) wherein
• a protein such as an antibody moiety containing a tyrosine hydroxy-aromatic ring (such as a tyrosine in an antibody such as ING-1).
• amine (85) can be treated with excess cyanuric chloride to produce the dichlorocyanuramide (88) and with 2-methoxycyanuric dichloride to produce the chloro-methoxycyanuramide (89). Both of these derivatives can be reacted at amine groups such as lysine amines or the terminal amine groups of peptide chains in proteins. Reaction occurs via the displacement of chloride from the triazine ring.
• N-hydroxysuccinimido ester of alpha-iodoacetic acid or with alpha-iodoacetic acid anhydride to produce the iodoacetamide derivative (90).
• This material can be reacted with proteins containing free sulfhydryl groups such as those utilized in the reactions of maleimide derivative (84c) above.
• lodoacetamide (90) will also react with amines on proteins, such as the epsilon amine of lysine and the terminal amine of a peptide chain.
• amine (85) can be alkylated with nitrile-containing alkylhalides such as Br-(CH 2 ) n -CN to
• Amine (85) can also be alkylated with nitrile-containing benzylic halides such as alpha-bromomethylbenzonitriles to provide nitriles (92).
• amine (85) can be acylated with active esters (or similarly activated carbonyl
• nitrile amides 93) and (94). Any of these nitriles can be converted into the corresponding amidates by the action of alcohol and anhydrous HCl to provide (95), (96), (97), and (98), respectively.
• These amidates will react with amines on proteins such as the epsilon amines of lysines and the terminal amines of peptide chains.
• iminodiacetic acid moieties can be protonated or can be present as ionic species (represented, for example, as sodium salts).
• the extent of protonation and of deprotonation of any one species is a function of the pH of the reaction medium which contains both the protein reactive group containing reagent and the protein.
• buffer salts such as, for example, phosphate, borate, citrate and acetate buffers to control the pH of the reaction medium is a part of this aspect of the invention.
• proteins may be subsequently modified with agents such as PEG (polyethylene glycol) reagents as is well known in the art to impart reduced immunogenicity to the modified proteins.
• agents such as PEG (polyethylene glycol) reagents as is well known in the art to impart reduced immunogenicity to the modified proteins.
• the modified proteins can be treated with radioisotopes of metal ions such as 90 Y +3 (as a non-limiting example) and the radionuclide-chelate-protein can be used for the therapeutic treatment of tumors, particularly if the protein is a tumor antigen specific antibody or fragment thereof.
• radioisotopes of metal ions such as 90 Y +3 (as a non-limiting example)
• the radionuclide-chelate-protein can be used for the therapeutic treatment of tumors, particularly if the protein is a tumor antigen specific antibody or fragment thereof.
• the modified proteins can be treated with radioisotopes of metal ions such as 111 In +3 or 187 Y +3 (as non-limiting examples) and the radionuclide-chelate-protein so produced can be used for the diagnostic imaging of tumors in cancer
• the protein is a tumor antigen specific antibody or fragment thereof.
• novel terpyridines and phenanthrolines of this invention can be prepared according to the above described synthetic techniques. Additional illustrative preparations are set forth in the examples which follow.
• the targeting radioactive immunoreagent of this invention is set forth in the examples which follow.
• the radionuclide ion can be selected, for example, from Sc, Fe, Pb, Ga, Y, Bi, Mn, Cu, Cr, Zn, Ge, Mo, Tc, Ru, In, Sn, Sm, Sb, W, Re, Po, Ta and Tl ions.
• Preferred radionuclides include 44 Sc +++ , 64,67 Cu ++ , 111 In +++ , 212P b ++ , 68 Ga ++ , 90 Y +++ and 212 Bi +++ ions. Of these, the most preferred are 90 Y +++ ions.
• the metal radionuclide ion and the complexing agent are easily complexed by merely mixing an aqueous solution of the complexing agent with a metal radionuclide salt in an aqueous solution preferably having a pH of 4 to 11.
• the salt can be any water soluble salt of the metal such as halogen salts.
• the chelate is generally prepared in aqueous solution at a pH of between 5 and 9 and preferably from 6 to 8.
• the complex optionally is mixed with buffers such as acetate, phosphate and borate to produce the optimum pH.
• the targeting immunoreagent of this invention includes an immunoreactive group covalently bonded to the complexing agent.
• the targeting immunoreagent thus comprises a conjugate of a complex having the structure A-I above and the
• immunoreactive group The complexing agent and the metal radionuclide can be complexed either before or after the complexing agent is attached to the immunoreactive group.
• immunoreactive group is meant to include any organic compound which is capable of covalently bonding to the complexing agent and which is found in a living organism or is useful in the diagnosis, treatment or genetic engineering of cellular material or living organisms, and which has a capacity for interaction with another component which may be found in biological fluids or associated with cells to be treated such as tumor cells.
• the immunoreactive group can be selected from a wide variety of naturally
• triiodothyronine gentamicin, carbamazepine, and theophylline
• steroids vitamins, polysaccharides, viruses, protozoa, fungi, parasites, rickettsia, molds, and components thereof, blood components, tissue and organ components, pharmaceuticals, haptens, lectins, toxins, nucleic acids (including
• oligonucleotides include antibodies (including genetically engineered antibodies and fragments thereof), antibody fragments,
• antigenic materials including proteins and carbohydrates
• avidin and derivatives thereof include avidin and derivatives thereof, biotin and derivatives thereof, and others known to one skilled in the art.
• Preferred immunoreactive groups for use in the practice of this invention are those which have a receptor molecule specific to a ligand of interest.
• a specific binding reaction involving the reagent can be used for the targeting expected.
• ligand-receptor complexes include, but are not limited to antibody-antigen, avidin-biotin, repressor (inducer) - promoter of operons and sugar-lectin complexes.
• complementary nucleic acids i.e., a hybridized product of complementary strands, are also
• immunoreactive groups include
• the immunoreactive group can be an antigenic material, an antibody, or an anti-antibody. Both monoclonal and polyclonal antibodies are useful.
• the antibodies can be whole molecules or various fragments thereof, as long as they contain at least one reactive site for reaction with the reactive groups on the complexing agent or with linking groups as described herein.
• Specifically contemplated as being included within these preferred immunoreactive groups are antibodies and proteins produced by the techniques of molecular biology.
• the immunoreactive group can be an enzyme which has a reactive group for attachment to the complexing agent.
• Representative enzymes include, but are not limited to, aspartate aminotransaminase, alanine
• the immunoreactive group can be modified or chemically altered to provide reactive groups for attaching to the complexing agent by techniques known to those skilled in the art. Such techniques include the use of linking moieties and chemical modification such as described in WO-A-89/02931 and WO-A-89/02932, which are directed to modification of oligonucleotides, and U.S. Patent 4,719,182 the disclosures of all of which are hereby incorporated herein by reference in their entirety. Two highly preferred uses of the targeting
• immunoreagents of this invention are for the diagnostic imaging of tumors and the radiological treatment of tumors.
• Preferred immunological groups therefore include antibodies to tumor-associated antigens. Specific examples include B72.3
• Biomira, Inc. of Edmonton, Canada which recognize squamous cell carcinomas, B43 antibodies which are reactive with certain lymphomas and leukemias and others which may be of particular interest.
• immunoreactive group is intended to include immunological groups having complexing agent molecules bonded thereto through one or more protein reactive groups.
• containing a carbohydrate region can be attached to the
• protein reactive group as defined herein is intended to include such linkages.
• Other techniques for performing the covalent binding of the immunoreactive group to the radioactive metal complexing agents are known in the art and include simply mixing the materials together.
• the radioactive immunoreagent of this invention can contain any ratio of metal radionuclide ion to complexing agent.
• the mole ratio of metal ion to complexing agent is from about 1:100 to about 1:1.
• the ratio of the complexing agent to the immunoreactive group can vary widely from about 0.5:1 to 10 : 1 or more. In some embodiments, the mole ratio of complexing agent to immunoreactive groups is from about 1 : 1 to about 6:1.
• Figs. 6 and 7 are absorption spectra of complexing agents and metal complexes of this invention. Portions of the spectra do not overlap with those of the proteins to which the complexing agents are chemically bonded. Similar spectral shifts were obtained between complexing agents of this
• the immunoreagent of this invention can be readily spectrophotometrically analyzed.
• the following examples further illustrate the
• 2-Acetyl-6-bromopyridine, 60 was synthesized by the method of J.E. Parks, B.E. Wagner, and R.E. Holm, J.
• p-anisaldehyde were dissolved together in 400 mL of methanol, and the solution was poured into the KOH solution.
• Nitrotetraester 68 (1.80 g, 2.29 mmol) was dissolved in a mixture of 90 mL of THF and 90 mL of abs. EtOH. Ammonium formate (2.89 g, 45.8 mmol) dissolved in 16 mL of H 2 O was added, followed by 4.8 g of 10% Pd/C (4.6 mmol). After stirring at room temperature for 2 h, the reaction was filtered through a diatomaceous earth filter pad. The filter pad was washed well with THF, abs. EtOH, and CH 2 Cl 2 . The filtrate was concentrated, and the residue was partitioned between CH 2 Cl 2 and aqueous NaCl.
• Amine tetraester 69 (0.75 g, 0.99 mmol) was stirred with 4 equiv of NaOH in a mixture of 50 mL of MeOH and 2 mL of H 2 O for 16 hours at room temperature. The mixture was
• Step 1 2-Acetyl-6-bromopyridine, 60 A 5 L 3-neck flask was charged with 2,6-dibromopyridine
• Step 2 1-[2-(6-Bromo-2-pyridinyl)-2-oxoethyl]pyridiniumiodide
• Step 5 4'-(4-methoxyphenyl)-2,2':6'2"-terpyridine-6,6"-dicarboxaldehyde
• a 3 L flask was charged with 400 mL THF and, while stirring under nitrogen, was cooled in a dry ice/ether bath to -78°C.
• a solution of n-butyllithium (55 mL; 2.5 M) was transferred via nitrogen pump to a pressure equalizing dropping funnel and added to the THF solution. After stirring 15 minutes, the temperature of the mixture was at -78°C and the solid bis-bromide (product of Step 4) (21 g; 42 mole) was added (Gooch tube) portion wise over an 11 minute period. The temperature did not exceed -75°C during the addition.
• Step 5 To stirred suspension of the bis-aldehyde from Step 5 (18.5 g; 46.8 mmoles) in 250 mL absolute ethanol at room temperature was added, portion wise, NaBH 4 (2.3 g; 60.8 mmoles) over about 10 minutes. Refluxing the mixture for 15 minutes gave a yellow solution, which was stirred for an additional 30 minutes and then diluted with 150 mL H 2 O. A precipitate developed. After stirring for one and one-half hours, the mixture was filtered. The light tan crystals, after drying, weighted 16 g (86% yield). The crude crystalline product was used without further purification in the next step. MP 140-141°C. Step 7 - Bismesylate of terpyridine of Step 6
• the pooled material was purified by flash chromatography using a column that was packed (4.5" wide ⁇ 2.5" high) with silica gel 60 (EM Science) under nitrogen with hexane containing 1%
• the bis (methylhydrazine) terpyridine of Part A (3.50 g, 82 mmol), ethyl bromoacetate (13.2 mL, 820 mmol), 2, 6-lutidine (9.6 mL, 820 mmol), and sodium iodide (0.35 g, 2 mmol) were added to 350 mL of acetonitrile, and the solution was refluxed under N2 for 48 hours, when an additional 4.1 mL (370 mmol) of ethyl bromoacetate and 4.8 mL (410 mmol) of 2,6-lutidine were added. The reaction solution was refluxed for an additional 48 hours, and cooled. A copious amount of white salt resulting from excess bromoacetate and lutidine was filtered and
• the terpyridine tetraester of Part B (0.659 g, 0.84 mmol) was dissolved in 5 mL of cone H 2 SO 4 to give a red-orange solution.
• the mixture was cooled to 0°C, and a mixture of cone HNO 3 in cone H 2 SO 4 was added such that 0.84 mmol of HNO 3 was delivered.
• the color of the solution turned pale yellow after addition was completed.
• the reaction mixture was stirred for 15 minutes at 0°C, then poured onto crushed ice. Dilute K 2 CO 3 was added until pH 8 was reached.
• the aqueous solution was extracted three times with CH 2 Cl 2 .
• Part D 4'-(3-Amino-4-methoxyphenyl)-6,6"-bis(N',N'- di(ethoxycarbonylmethyl)-N-methylhydrazino)- 2,2':6',2"-terpyridine
• the nitrotetraester of Part C (0.72 g, 0.88 mmol) was dissolved in a mixture of 30 mL of THF and 30 mL of ethanol.
• Ammonium formate (1.08 g, 17.1 mmol) dissolved in 6 mL H 2 O was added, followed by 1.8 g of 10% Pd/C (1.7 mmol).
• the THT amine tetrasodium salt of Preparation 2, Part E (0.39 g, 0.51 mmol) was dissolved in 80 mL of methanol. At room temperature, 0.58 g (0.50 mmol) of thiophosgene in 1.0 mL tetrahydrofuran was added, followed by addition of 0.51 g (0.50 mmol) of triethylamine in 1.0 mL of tetrahydrofuran. The reaction was then concentrated to a residue, which was slurried in dichloromethane, and filtered to yield 0.26 g (65%) of yellow solid. The infrared spectrum was consistent with the assigned structure. The product was believed to be the
• Neocuproine hydrochloride hemihydrate (25.0 g, 99 mmol) was dissolved in 100 mL of concentrated nitric acid. 200 mL of concentrated sulfuric acid was added, and the reaction was heated at reflux for 2.5 hours, and then allowed to stand at room temperature for 8 days. The reaction mixture was then gradually added to a mixture of about. 3 Kg of ice and 351 g (8.8 mole) of LiOH ⁇ H 2 O, while stirring with a glass rod.
• the reaction mixture was partitioned between 400 mL of diethyl ether and 400 mL of distilled H 2 O and the ether fraction was extracted a total of 5 times with 400 mL portions of distilled H 2 O.
• the ether phase was concentrated and dissolved in 400 mL of methylene chloride.
• the methylene chloride phase was extracted once with distilled H 2 O, dried with a mixture of
• Tetrasodium Salt TMT, 70, of Preparation 1 (0.31 g, 0.42 mmol) was dissolved in
• N-methylpyrrolidin-2-one containing 2 % by weight of 2,9-bisbromomethyl-5-nitro-1,10-phenanthroline prepared in Preparation 4, Part B is added via a syringe pump over 2 hours into 20 mL of magnetically stirred N-methylpyrrolidin-2-one which is held under argon at 20°C.
• a 2 % solution in N-methylpyrrolidin-2-one of one equivalent of the disodium salt of diethylenetriamine N,N',N"-tri-p-toluenesulfonamide is added.
• reaction is stirred for 6 additional hours after the addition at 20°C, 200 mL of the solvent is then distilled under high vacuum, the residual solution is cooled and added to 100 mL of ice water. The resulting precipitate is isolated by filtration, washed with cold water, and triturated with acetonitrile to provide the crude tri-p-toluenesulfonamide derivative.
• One part of crude macrocycle prepared in Part A is dissolved in 10 parts of concentrated sulfuric acid (96%), and the reaction mixture is stirred and heated to 110°C under argon. After 24 hours, the solution is cooled with vigorous stirring in an ice water bath and is treated with 10% sodium hydroxide solution until the pH reaches 10. The aqueous phase is extracted five times with equal volumes of chloroform, the extracts are combined and are dried over anhydrous sodium .sulfate. The salts are then removed by filtration, and the filtrate is evaporated under argon.
• the crude triamine can be purified by crystallization from ethanol/water.
• a mixture of one part of the triamino macrocycle prepared in Part B with 10 parts of sodium carbonate and 3 parts of ethyl bromoacetate as a 3 percent by weight solution in anhydrous acetonitrile is stirred under argon for 24 h at 60°C.
• the reaction mixture is then cooled to room temperature, filtered, and the solvent is evaporated. The residue can be further purified by trituration in cold ether.
• Preparation 7, Part E is stirred in 200 parts of methanol at room temperature. This is treated at ambient temperature with one equivalent of thiophosgene dissolved in two parts by weight of tetrahydrofuran followed by one equivalent of triethylamine dissolved in. two parts by weight of tetrahydrofuran. After one hour, the reaction mixture is concentrated to a residue, slurried in dichloromethane, and the product is isolated by filtration.
• dimethylformamide is allowed to react at 160°C for 6 h. After cooling, the reaction mixture was transferred into 1500 mL of water and the resulting mixture is stirred for 2 h and
• the organic layer is washed successively with water (2 ⁇ 30 mL), 0.01 N HCl solution (2 ⁇ 25 mL), water (1 ⁇ 20 mL), 5% NaHCO 3 solution (2 ⁇ 25 mL), and water (2 ⁇ 20 mL). After drying ( Na 2 SO 4 ), the solvent is distilled to yield the desired dilactam which is then purified by silica gel chromatography.
• the cyclic dilactam (a) of Part C above is reduced with diborane to provide the diamine which is reacted with ethyl bromoacetate to provide the bis(ethyl acetate). This is then nitrated with nitric acid and sulfuric acid to provide the 4'-(4-methoxy-3-nitrophenyl)-2,2':6'2"-terpyridinyl derivative.
• the nitro group is then reduced with ammonium formate in the presence of palladium on carbon, and the ester groups are saponified with sodium hydroxide to provide the desired macrocylic chelate (b).
• the tritoluenesulfonamide, 14(a), of Part A above is hydrolyzed in sulfuric acid to remove the toluenesulfonamide groups and provide the macrocyclic terpyridinyl triamine derivative.
• This triamine is reacted with ethyl bromoacetate to provide the tris (ethyl acetate).
• This is nitrated with nitric acid and sulfuric acid to provide the 4'-(4-methoxy-3-nitrophenyl)-2,2':6'2"-terpyridinyl derivative.
• TMT or THT was attached to antibodies by oxidation of the carbohydrate groups on the antibodies with NaIO 4 to produce aldehyde groups on the antibodies.
• a solution (110 mL) of 0.1 M NaIO 4 in purified water was added to one mL of a 4 mg/mL solution of B72.3* antibody in PBS (10 mM sodium phosphate, 0.15 M NaCl), pH 6.0.
• the pH of the resulting solution was readjusted to 6.0 and incubated for 1 hour at room temperature in the dark. After this incubation, excess periodate was removed by passing the antibody solution through a Sephadex G50 column that had been equilibrated with PBS, pH 6.0.
• the conjugate preparations produced by this procedure had an average molar ratio of TMT or THT to antibody of about 1.7.
• the concentrations of antibody in the conjugate solutions were determined using the BioRad protein assay using bovine immunoglobulin as the protein standard.
• THT concentration of THT was determined spectrophotometrically using the measured extinction coefficient at 350 nM
• concentration of THT was determined spectrophotometrically using the measured extinction coefficient at 350 nM
• TMT-B72.3 conjugate a portion (about 0.5 mg) of the conjugate was put into 1 mL of 0.01 M sodium acetate, 0.15 M NaCl buffer, pH 6.0 and an excess of Eu +3 was added. The absorbance of this solution at 330 nm was used to determine the amount of chelate in these solutions.
• Linbro EIA II Plus microtiter plates were coated with antigen by adding 100 mL/well of a solution of 4 mg/mL of bovine submaxillary mucin (Sigma M4503) , an antigen reactive with the B72 .3 antibody, in PBS (phosphate buffered saline) and incubated for 1 h at room temperature. After washing the plate three times with PBS, pH 6.8, the wells were blocked by adding 200 mL/well of a 1% BSA (bovine serum
• Figure 1 also contains the curve for a scandium complex of the B72.3-THT conjugate prepared in the same manner as the Europium complex described hereinbefore.
• the antibody-chelate conjugates to be labeled with either H ⁇ In or 90Y were in phosphate buffered saline, pH 6.0.
• the radionuclides (about 1 mCi) were added to the conjugate solution (1 mL containing about 1 mg of antibody-chelate conjugate), mixed, and incubated about 30 min at room temperature. Any non-chelated metal was removed from each conjugate preparation by HPLC gel filtration (TSK3000SW column). The column effluent was monitored for both protein (OD280 nm) and radioactivity (radioactivity monitor). The specific activity of the purified labeled conjugates was calculated from this data. The conjugates were used for biodistribution studies immediately.
• mice were used in each test group for the 90 Y biodistribution study and 3 mice in each group for the m-In study.
• Target doses for each mouse were 10 mg of radioactive conjugate per dose, i.e.'s 20-100 ⁇ g of antibody per ⁇ L, and greater than 50 ⁇ Ci per dose. Both non-tumor bearing and tumor bearing mice were injected on Day 0.
• mice were injected subcutaneously in the left rear flank with one million LS174T cells in exponential growth phase, in about 0.2 mL of sterile medium or saline from cell culture. The mice were examined for tumor growth until tumors became measurable. Thereafter, tumors were measured to the nearest 0.1 mm across two perpendicular diameters (length ⁇ width) using digital calipers until the product of the length times the width was between 50 and 100 square millimeters for all the mice.
• mice were anesthetized by injecting i.p. 0.15 mL of a sterile solution containing
• ketamine HCl at 11 mg/mL and xylazine at 3 mg/mL.
• the test material was injected via the retroorbital venous sinus. Each mouse was counted in the dose calibrator immediately after injection, the syringes were reweighed, recounted and the data recorded.
• each animal was counted in the dose calibrator following standard operating procedures. Each carcass was counted after dissection.
• each animal had its tumor measured to the nearest 0.1 mm across two perpendicular diameters (length ⁇ width) using digital calipers.
• a blood sample was collected from each animal into a tared culture tube and then the tube was reweighed.
• Each animal was sacrificed by cervical dislocation.
• Organs (lungs, spleen, liver, colon, right kidney, left kidney, tumor, bone marrow, muscle) were dissected and trimmed to remove extraneous tissue and then weighed to the . nearest milligram. Radioactivity of the dissected organs was determined by gamma counting.
• the biodistrubtions shown in Figure 4 are those obtained eight days after injection of the radioactive
• TMT is a better chelate for antibody targeted radionuclide therapy using this isotope.
• the blood was found to have a higher 90 Y level when TMT was the chelator used to prepare the immunoconjugate. This is believed to be due to superior in vivo stability of the B72.3-TMT- 90 Y complex compared to the B72.3-DTPA- 90 Y complex.
• mice innoculated with these conjugates should find the B72.3-TMT- 90 Y conjugate less toxic than those innoculated with B72.3-DTPA- 90 Y.
• the general procedures to test this hypothesis were the same as those described for the biodistribution study with the following exceptions. There were 8 tumor bearing nude mice in each test group.
• the injection schedule was as follows: 200 ⁇ Ci, 120 ⁇ Ci, and 120 ⁇ Ci were given on days 0, 4 and 8, respectively, for both the B72.3-TMT- 90 Y or B72.3-DTPA- 90 Y conjugate (low dose regimen). Two other sets of mice received 400 ⁇ Ci, 320 ⁇ Ci and 320 ⁇ Ci of the two
• TMT or a suitable derivative thereof can be conjugated to an antibody molecule to yield an antibody-TMT conjugate molecule that displays the ability to bind to a target antigen recognized by the antibody variable region.
• a conjugate molecule can be used to deliver a radioisotope that is chelated by the TMT moiety in order to localize and/or treat the lesion that is targeted by such an immunoconjugate.
• the antibody is selected such that it has a broad reactivity with an antigen molecule expressed on tumor cells, thereby providing an antibody-TMT conjugate that can deliver radioisotope to the tumors for therapeutic or diagnostic purposes.
• ING-1 is a chimeric antibody (described in
• the antibody molecule is produced by culturing a mouse myeloma cell line expressing the chimeric antibody essentially as described in the above-referenced publication.
• the chimeric ING-1 antibody is used at a concentration of 5.2 mg/mL in 50mM sodium acetate buffer at pH 5.6 and supplemented with 150mM NaCl.
• To 1.15 mL of antibody solution is added a solution of 50mM sodium borate at pH 9.0 supplemented with 100mM sodium chloride to a final total volume of 2.5 mL.
• the solution is applied to a PD-10
• TMT-NCS NCS derivative of TMT
• the solution of the NCS derivative of TMT is prepared in the sodium borate buffer at a concentration of 10mg/mL and added to the antibody solution to a final concentration of 138 ⁇ M of TMT-NCS.
• the solution is gently mixed and incubated in the dark at ambient temperature (approx. 22°C) overnight (approx. 12 hr).
• the ING-1:TMT conjugate is separated from free TMT-NCS and other low
• immunoconjugate is then tested for its ability to bind to the tumor cell antigen and also to bind Yttrium-90 isotope to demonstrate that the conjugate can be radiolabeled in the TMT moiety and can target the tumor cell.
• the antibody ING-1 was produced as above. After purification, ING-1 was used at a concentration of 5.0 mg/mL in 50 mM sodium acetate and 150 mM sodium chloride buffer, pH 5.6.
• the conjugation of ING-1 to TMT-NCS was achieved by first adding 1.0M carbonate, 150 mM sodium chloride buffer, pH 9.3 to ING-1 until the antibody solution reaches a pH of 9.0. A sample of that ING-1 solution containing 5 mg of protein was then pipetted into an acid washed, conical, glass reaction vial. A solution of TMT-NCS (Preparation 6) was prepared by dissolving 100 mg in 10 mL of 1.0 M carbonate, 150 mM sodium chloride buffer, ⁇ H9.3. The conjugation reaction was started by the addition of 96.5 ⁇ L of the TMT-NCS solution to the antibody solution to give a 4-fold molar excess of TMT-NCS over ING-1. The solution was stirred briefly to mix the reactants and then left was in the dark at room temperature. After 16 hours, the ING-1 conjugate with TMT was separated from
• the concentrations of ING-1 in the conjugate solutions were determined by the BioRad protein assay using bovine immunoglobulin as the protein standard.
• ING-1/TMT europium chloride
• the antigen to which the antibody, ING-1, binds was prepared from LS174T or HT 29 cells (available from ATTC) by scraping confluent monolayers of cells from the walls of culture flasks with a cell scraper. The cells from many flasks were combined and a sample was taken and counted to estimate the total number of cells harvested. At all times the cells were kept on ice. Following centrifugation of the cells at 1500 rpm for 10 minutes at 4°C , the cells were washed once in ice-cold 50mM sodium phosphate buffer, pH 7.4 supplemented with 150 mM sodium chloride (PBS), pelleted under the same
• the cells were homogenized at 4°C using a motor-driven pestle and then centrifuged at 3000 ⁇ g for 5 minutes. The antigen-rich supernatant was removed from the other cell debris and
• the pellet (antigen fraction) from this final step was suspended in 100 ⁇ L of PBS for every million cells harvested. Following an estimate of the protein concentration (BioRad BCA protein assay using bovine immunoglobulin as the protein standard) the antigen was stored at at -20°C until use.
• Each well of a 96-well Costar microtiter plates was coated with antigen by adding 100 ⁇ L/well of cell lysate (10 ⁇ g/mL) prepared as described above.
• the microtitre plates were allowed to dry overnight in a 37°C incubator. After washing the plate five times with 0.05% Tween-20 (Sigma), the wells were blocked by adding 125 ⁇ L/well of a 1% BSA (bovine serum albumin, Sigma A-7906) solution in PBS and incubated for 1 h at room temperature. The plates were washed five times with 0.05% Tween-20.
• BSA bovine serum albumin
• phosphatase substrate reagent two Sigma 104 phosphatase tablets dissolved in 10 mL distilled water and 20 ⁇ L Sigma 221 alkaline buffer. After one hour at room temperature, the color was read using a 405 nm filter in a Titertek Multiscan microplate reader.
• HT29 cells were grown to confluency in tissue culture flasks using DMEM media supplemented with 10% fetal calf serum. The cells were harvested by scraping the flask walls with a cell scraper. Cells from many separate flasks were pooled, centrifuged to a pellet, resuspended at 5x10 5 /mL in a solution of ice-cold 50mM sodium phosphate with 150 mM sodium chloride buffer pH 7.4 (PBS) supplemented with 0.1% bovine serum albumin (Sigma) and 0.02% sodium azide (Flow buffer). The cells were washed in this same buffer and then counted.
• PBS sodium chloride buffer pH 7.4
• Flow buffer 0.1% bovine serum albumin
• An antibody standard curve was constructed by diluting ING-1 with an irrelevant (non binding), isotype-matched control antibody (human IgG1) to give a number of samples ranging in ING-1 content from 10% to 100%.
• the standard curve was made in flow buffer so that each sample contained 1.0 ⁇ g protein per mL. Samples from the standard curve and unknowns were then incubated with 5x10 5 HT29 cells at 4°C for 1 hour. After extensive washing to remove unbound antibody, the cells were resuspended in 100 ⁇ L flow buffer and incubated at 4°C for 1 hour with goat-anti-human antibody labelled with fluorescene isothiocyanate (FITC).
• FITC fluorescene isothiocyanate
• Analysis windows were applied to these parameters to separate single cells from aggregates and cell debris. Fluorescence from FITC and propidium were separated with a 550nm long pass dichroic filter and collected through a 530 nm band pass filter (for FITC), and a 635 nm band pass filter (for PI). Light scatter parameters were collected as integrated pulses and fluorescence was collected as log integrated pulses. Dead .cells were excluded from the assay by placing an analysis window on cells negative for PI uptake. The mean fluorescence per sample (weighted average from 2500 cells) was calculated for each histogram. FITC calibration beads were analysed in each experiment to establish a standard curve. The average fluorescence intensity for each sample was then expressed as the average FITC equivalents per cell. Immunoreactivity was calculated by comparing the average fluorescence intensity of the unknown sample with values from the standard curve.
• ING-1/TMT had a major peak also at 9.1 minutes but a minor peak, attributable to aggregates, was sometimes seen at 7.3 minutes. By comparison of the peak areas, the aggregate peak was less than 5% of the total.
• Radiolabeling of ING-1/TMT with 90 Y A volume of radioactive yttrium chloride ( 90 Y in 0.04M hydrochloric acid at a specific activity of >500 Ci/g:
• the strip was inserted into a System 200 Imaging Scanner (Bioscan) which had been optimized for 90 Y and which was controlled by a Compaq 386/20e computer. In this system free 90 Y migrates at the solvent front while the ING-1/TMT ( 90 Y) remains at the origin.
• Bioscan System 200 Imaging Scanner
• Binding of lanthanides such as europium (III) to chelating agents that contain an aromatic moiety held close to the co-ordination sphere can lead to "sensitized” fluorescence wherein light is absorbed through the aromatic system and the energy is transfered to the metal. The metal then produces emissions characterized by a very large Stokes shift and fluorescence lifetimes of up to several seconds.
• a 0.5 mg aliquot of the ING-1/TMT in 2.5 mL in 0.05 M Tris HCl buffer pH 7.5 was pipetted into a 4 mL conical reaction vial containing a small stirring bar.
• a 250 ⁇ M europium chloride (europium chloride hexahydrate: Aldrich) solution in 0.05 M Tris HCl buffer pH 7.5 was prepared. An aliquot (50 ⁇ L) of this europium chloride solution was added to the reaction vial containing ING-1/TMT, and the resulting solution was stirred very slowly on a magnetic stirrer at room temperature. The labelling was allowed to proceed for one hour and then the reaction mixture was loaded on to a PD-10 chromatography column which had been pre-washed and equilibrated in a buffer
• PR1A3 is a murine antibody (Developed by ICRF, U.K.) The antibody was used at a concentration of 4.0 mg/mL in 50mM sodium acetate buffer at pH 5.6 and supplemented with 150mM
• TMT-NCS TMT-NCS.
• the solution was gently mixed and incubated in the dark at ambient temperature (approx. 22°C) overnight (14-16 hr).
• the PR1A3:TMT conjugate was separated from free TMT-NCS and other low molecular weight products using a Superose 6 HPLC column equilibrated and eluted in 50mM sodium phosphate buffer at pH 7.2 supplemented with 150mM sodium chloride.
• the eluate was concentrated using a Centricon-30 ultrafiltration device to a concentration of 0.25 mg PR1A3:TMT per milliliter solution.
• the TMT immunoconjugate is then tested for its ability to bind to the tumor cell antigen and also to bind yttrium-90 isotope.
• SW 1222 cells were used to test the immunoreactivity using flow cytometry. These cells (ATCC) were grown in MEM medium supplemented with 10% fetal calf serum and non-essential amino acids in Earles balanced salt solution. ELISA tests for immunoreactivity were not performed on PR1A3/TMT conjugates.
• NRLU-10 is a murine antibody (Developed by Okabe as TFS-2).
• the antibody was used at a concentration of 3.4 mg/mL in 50mM sodium phosphate buffer at pH 7.2 and supplemented with 150mM NaCl.
• To 12 mg of antibody solution was added 1.5 mL of a solution of 50mM sodium borate at pH 9.0 supplemented with 150mM sodium chloride.
• Two aliquots (2.5 mL each) of that solution were applied to separate PD-10 chromatography columns equilibrated with 50mM sodium borate at pH 9.0 supplemented with 150mM sodium chloride.
• the antibody was eluted off each column with 3.5 mL of the same sodium borate buffer. These eluates were pooled.
• TMT-NCS TMT-NCS
• sodium borate buffer pH 8.3
• 19 microliters was added to the antibody preparation.
• the solution was gently mixed and incubated in the dark at ambient temperature (approx. 22°C) overnight (approx. 12 hr).
• NRLU-10:TMT conjugate was separated from free TMT-NCS and other low molecular weight products using a Superose 12 HPLC column equilibrated and eluted in 50mM sodium phosphate buffer at pH 6.0 supplemented with 150mM sodium chloride.
• immunoconjugate was then tested for its ability to bind to the tumor cell antigen and also to bind yttrium-90 isotope.
• Example 6 Preparation of an immunoreactive agent comprising of an oligonucleotide covalently bonded to the complexing agent.
• the complexing agent TMT can be covalently linked to an immunoreactive group other than an antibody, for example an oligonucleotide.
• This immunoreagent can be labelled with 90 Y and the radioactive immunoreagent thus prepared can react with its compliment, namely the complimentary oligonucleotide.
• a 20-mer oligonucleotide containing a 3'-amine group, 5'-TTAGCTTCTCCGTCCATAA-C 5 Amine-T-3', cl, is prepared on an Applied Biosystems DNA synthesizer as directed by the equipment manufacturer using Uni-link Amino Modifier (Clonetech) as the precursor to the 3'-amine group and the 2-deoxynucleotide phosphoramidite reagent precursors 5'-dimethoxytrityl cytidine-3'-O-phosphoramidite, 5'-dimethoxytrityl adenosine-3'-O-phosphoramidite, 5'-dimethoxytrityl guanosine-3'-O-phosphoramidite, and 5'-dimethoxytrityl thymidine-3'-O-phosphoramidite (Applied Biosystems).
• the amine-functionalized oligonucleotide is further purified by elution down an OPEC Cantridge (Polymer RPI) column (Clonetech) with deionized water. The concentration of oligonucleotide is monitored using absorbance at 260 nm.
• Part B Preparation of an oligonucleotide conjugated to TMT, cI-TMT.
• a 20-mer oligonucleotide containing a 3'-amine group 5'-TCT TAT GGA CGG AGA AGC-C5 Amine-T-3, is prepared on an
• the amine-functionalized oligonucleotide is further purified by elution down an OPEC cartridge (Polymer RPI) column (Clonetech) with deionized water.
• OPEC cartridge Polymer RPI
• oligonucleotide I prepared in part D in phosphate buffered saline, pH 7.4, at 4°C is added a solution of the 90 Y-labeled TMC-cI as described above. After 1 hour, the components of the reaction mixture are analyzed by polyacrylamide gel electrophoresis. The radiolabel is observed at the position in the gel corresponding to the hybridized oligonucleotide.

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• 1992
  • 1992-05-07 AU AU23161/92A patent/AU672951B2/en not_active Expired
  • 1992-05-07 JP JP51920993A patent/JP3358141B2/ja not_active Expired - Lifetime
  • 1992-05-07 EP EP92915130A patent/EP0639083B1/de not_active Expired - Lifetime
  • 1992-05-07 KR KR1019940703965A patent/KR950701229A/ko not_active Abandoned
  • 1992-05-07 DE DE69231952T patent/DE69231952T2/de not_active Expired - Lifetime
  • 1992-05-07 BR BR9207126A patent/BR9207126A/pt not_active Application Discontinuation
  • 1992-05-07 CA CA002135059A patent/CA2135059A1/en not_active Abandoned
  • 1992-05-07 WO PCT/US1992/003858 patent/WO1993021957A1/en not_active Ceased
  • 1992-05-07 RU RU94046010A patent/RU2122431C1/ru active
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• 1994
  • 1994-11-02 NO NO944182A patent/NO944182L/no not_active Application Discontinuation
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